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WO1989005345A1 - Modification genetique de cellules endotheliales - Google Patents

Modification genetique de cellules endotheliales Download PDF

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Publication number
WO1989005345A1
WO1989005345A1 PCT/US1988/004383 US8804383W WO8905345A1 WO 1989005345 A1 WO1989005345 A1 WO 1989005345A1 US 8804383 W US8804383 W US 8804383W WO 8905345 A1 WO8905345 A1 WO 8905345A1
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Prior art keywords
endothelial cells
genetic material
cells
dna
interest
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PCT/US1988/004383
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English (en)
Inventor
James M. Wilson
Richard C. Mulligan
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Howard Hughes Medical Institute
Whitehead Institute for Biomedical Research
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Howard Hughes Medical Institute
Whitehead Institute for Biomedical Research
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Priority to EP89900771A priority Critical patent/EP0391960B1/fr
Priority to DE3851153T priority patent/DE3851153T2/de
Priority to HK98107130A priority patent/HK1008400A1/en
Publication of WO1989005345A1 publication Critical patent/WO1989005345A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/44—Vessels; Vascular smooth muscle cells; Endothelial cells; Endothelial progenitor cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/29—Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/069—Vascular Endothelial cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01023—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the endothelium is a single layer of flattened transparent cells which are joined edge to edge, with the result that they form a membrane of cells.
  • Endothelial cells originate during development from the embryonic mesoblast or mesoder . They occur on the free surfaces of serous membranes, in the anterior chamber of the eye and on the surface of the brain and spinal cord. In addition, they form the lining membrane of the heart, blood vessels and lymphatics.
  • the invention described herein relates to genetically engineered endothelial cells and particularly to genetically engineered endothelial cells which express selected genetic material of interest (DNA or RNA) which has been incorporated into them, for example by means of a retroviral vector having a recombinant genome which includes the genetic material of interest. It also relates to methods of stably introducing into endothelial cells such genetic material and methods of using the genetically engineered endothelial cells.
  • DNA or RNA selected genetic material of interest
  • Endothelial cells of this invention have stably incorporated in them genetic material of interest, which encodes a product (e.g., a protein or polypeptide) whose production in the endothelial cells is desired.
  • the modified endothelial cells express the incorporated genetic material (produce the encoded product) .
  • This genetic material of interest is referred to herein as incorporated genetic material.
  • the incorporated genetic material can be any selected DNA of interest (e.g., all or a portion of a gene endocing a product of interest) or RNA of interest.
  • DNA or RNA which is present in and expressed by normal endothelial cells DNA or ENA which does not normally occur in endothelial cells; DNA or RNA which normally occurs in endothelial cells but is not expressed in them at levels which are biologically significant (i.e., levels sufficient to produce the normal physiological effects of the protein or the polypeptide it encodes) ; DNA or RNA which occurs in endothelial cells and has been modified so that it is expressed in endothelial cells; and any DNA or RNA which can be modified to be expressed in endo ⁇ thelial cells, alone or in any combination thereof.
  • Endothelial cells of the present invention can also express genetic material encoding a selectable marker, thus providing a means by which cells expressing the incorporated genetic material are identified and selected for.
  • Endothelial cells containing incorporated genetic material are referred to as transduced endothelial cells.
  • retroviral vectors have been used to stably transduce endothelial cells with genetic material which includes genetic material encoding a polypeptide or protein of interest not normally expressed at biologically significant levels in endothelial cells.
  • the genetic material introduced in this manner also included genetic material encoding a dominant selectable marker.
  • Genetic material including DNA encoding a poly ⁇ peptide of interest and DNA encoding a dominant selectable marker has been introduced into cultured endothelial cells. Expression of these genes by the endothelial cells into which they have been incorporated (i.e., endothelial cells transduced by the use of retroviral vectors) has also been demonstrated.
  • transduced endothelial cells which express the incorporated genetic material they contain is also a subject of the present invention.
  • Transduced endothelial cells of the present invention are - A-
  • prosthetic implants e.g., vessels transplanted from one site in an individual to another; vessels implanted from another animal, such as pig, into an individual; vessels made of synthetic materials such as Dacron and polytetra- fluoroethylene, which are used in vascular reconstructive surgery
  • polypeptides or proteins useful in prevention and therapy or treatment, which are presently administered parenterally.
  • endothelial cells of the present invention can be designed to improve the characteristics of endothelial cell-lined prosthetic implants by enhancing or improving the ability of endothelial cells to seed or bind to the inner surface of the implant; by modifying the endothelial cells used to line an implant so that they will grow; or by overcoming the problem, encountered wi-th presently- available implants, of smooth muscle cell growth at the implant ends, which results in narrowing and, ultimately, closing off of the ends.
  • the transduced endothelial cells of the present invention can be used to provide a drug, hormone or enzyme delivery system.
  • prosthetic arterial grafts are often used to replace diseased arteries which perfuse vital organs or limbs.
  • the currently available grafts are usually made of synthetic material and are subject to many complications, the worst of which is a high rate of re-stenosis or occlusion.
  • Animal studies suggest that lining the graft with autologous endothelial cells prior to implantation may decrease, but not prevent, graft reocclusion with its attendant morbid consequences.
  • Endothelial cells can be modified according to the method of the present invention in a way that improves their performance in the context of an implanted graft.
  • Examples include secretion or expression of a transmembrane thrombolytic agent to prevent intraluminal clot formation, secretion of an inhibitor of smooth muscle proliferation to prevent luminal stenosis due to smooth muscle hypertrophy, and expression and/or secretion of an endothelial cell mitogen or autocrine factor to stimulate endothelial cell proliferation and improve the extent of rate of or duration of the endothelial cell lining of the graft lumen.
  • An additional application is to cover the surface of prosthetic heart valves with transduced endothelial cells in an attempt to improve the performance/longevity of the artificial valves.
  • a vascular implant lined with endothelial cells which include incorporated , genetic material encoding a polypeptide of interest synthesizes the encoded polypeptide and thus serves as a continuous delivery system for that polypeptide. In this way, the hormone or other polypeptide is secreted directly into the blood ⁇ stream of the individual receiving the implant.
  • endothelial cells of the present invention can be used to administer therapeutic proteins (e.g., hormones, enzymes, clotting factors) which are presently administered intravenously, intramuscularly or subcutaneously.
  • therapeutic proteins e.g., hormones, enzymes, clotting factors
  • endothelial cells modified according to the present invention produce the polypeptide hormone as it would normally be produced.
  • Another advantage to the use of genetically engineered endothelial cells lining prosthetic arterial implants is that one can target the delivery of therapeutic levels of a secreted product to a specific organ or limb.
  • the secreted product will be delivered to tissue perfused by. the grafted artery in high concentrations, thereby achieving a desired effect to a targeted anatomical location.
  • This product will then be diluted to nontherapeutic levels " in the venous circulation during its return to the heart.
  • one or more vessels containing transduced endothelial cells of the present invention which produce an angiogenic factor can be implanted into the leg of an individual whose circulation is compromised (e.g., as a complication of diabetes mellitus) resulting in inadequate blood flow to the foot.
  • HGH human growth hormone
  • genes can be introduced into endothelial cells using a retroviral vector, they can be "on" (subject to) the retroviral vector control; in such a case, the gene of interest is transcribed from a retroviral promoter.
  • a promoter is a specific nucleotide sequence recognized by RNA polymerase molecules that start RNA synthesis.
  • retroviral vectors having additional promoter elements ⁇ in addition to the promoter incorporated in the recombinant retrovirus) which are responsible for the transcription of the genetic material of interest can be used.
  • a construct in which there is an additional promoter modulated by an external factor or cue can be used, making it possible to control the level of polypeptide being produced by the endothelial cells by activating
  • heat shock proteins are proteins encoded by genes in which the promoter is regulated by temperature.
  • the promoter of the gene which encodes the metal-containing protein is regulated by temperature.
  • metallothionine is responsive to cadmium (Cd ) ions.
  • Figure 1 is a schematic representation of a wild type murine leukemia virus (retroviral) genome.
  • Figure 2 is a schematic representation of retroviral vectors, each having a recombinant genome, useful in the present invention.
  • Figure 2a is pLJ and
  • Figure 2b is pEm.
  • Figure 3 is a schematic representation of the construction of a recombinant retroviral vector, using the pLJ vector represented in Figure 2a and the human parathyroid hormone gene.
  • Figure 4 includes pictures of bovine aortic endothelial cells infected with a retrovirus that expresses low density lipoprotein receptor (LDLR) and analyzed for the uptake of fluorescent labeled LDL.
  • Panel A uninfected bovine aortic endothelial cells under phase contrast
  • Panel B uninfected bovine aortic endothelial cells under fluorescent illumination
  • Panel C infected bovine aortic endothelial cells under phase contrast
  • Panel D - infected bovine aortic endothelial cells under fluorescent- 1 illumination.
  • LDLR low density lipoprotein receptor
  • Figure 5 includes pictures of bovine aortic endothelial cells infected with a retrovirus that expresses B-galactosidase and analyzed for its expression using an in situ histochemical stain.
  • Panel A un ⁇ infected bovine aortic endothelial cells stained for beta-galactosidase activity
  • Panel B infected unselected bovine aortic endothelial cells stained for beta-galactosidase activity.
  • Genetic material of interest has been incorporated into endothelial-cells and expressed in the resulting genetically engineered endothelial cells.
  • Genetic material of interest incorporated into endothelial cells according to the method described can be any selected DNA of interest (e.g., all or a portion of a gene encoding a product of interest) or any selected RNA of interest.
  • it can be DNA or RNA which is present and expressed in normal endothelial cells; DNA or RNA which does not normally occur in endothelial cells; DNA or RNA which normally occurs in endothelial cells but is not expressed in them at levels which are biologically significant (levels sufficient to produce the normal physiological effects of the protein or polypeptide it encodes) ; DNA or RNA which occurs in endothelial cells and has been modified in such a manner that it can be expressed in such cells; and any DNA or RNA which can be modified to be expressed in endothelial cells, alone or in any combination thereof.
  • This genetic material of interest is referred to herein as incorporated genetic material.
  • Endothelial cells of the present invention express the incorporated genetic material ⁇
  • the incorporated genetic material (i.e., DNA or RNA) expressed by endothelial cells of the present invention is genetic material encoding a polypeptide or a protein of interest (genetic material of interest) .
  • Endothelial cells of the present invention can also include and express a gene encoding a selectable marker.
  • genetic material of interest which is DNA normally present in and expressed by human endothelial cells can be incorporated into abnormal endothelial cells (e.g., which contain an abnormal quantity of the DNA or which contain but do not express such material adequately) , with the result that they are able to produce the desired protein or polypeptide.
  • genetic material encoding a hormone has been introduced into endothelial cells by exposing them to media that contains a virus having a recombinant genome (i.e., by infecting them) .
  • the media used is obtained by harvesting media in which producer cells (e.g. , a Psi am or amphotropic producer) have been grown. That is, producer cells have been grown in tissue culture to a confluent density in Dulbecco's Modified Eagle's medium (DME) with 10% calf serum (CS) and penicillin and streptomycin. Fresh media' is added and subsequently (e.g., approximately 12 hours later), the media is harvested.
  • DME Dulbecco's Modified Eagle's medium
  • CS calf serum
  • Fresh media' is added and subsequently (e.g., approximately 12 hours later), the media is harvested.
  • Approximately 10 ml of media is harvested from a 10 cm plate of confluent producer cells.
  • the spent media (or. viral stock) is filtered through a 0.45 micron Millipore filter to remove detached producer cells and is used immediately to infect cells or is stored at -70 C.
  • Media is removed from a subconfluent plate of endothelial cells (recipient endothelial cells) and quickly replaced with viral stock (e.g., 5 ml/10 cm. plate) containing 8 mcg/ml. of Polybrene (Aldrich) . Subsequently (e.g., approximately 12 hours later), this is removed and replaced with fresh media.
  • the media used is a viral supernatant.
  • the recombinant genome of the infectious virus includes the genetic material of interest, which is incorporated into endothelial cells.
  • the recombinant genome can also have genetic material encoding a dominant selectable marker.
  • endothelial cells were made which express a polypeptide not normally expressed by such cells at biologically significant levels and, optionally, a dominant selectable marker.
  • endothelial cells are exposed to media containing infectious virus produced in Psi am cells; the infectious virus contain a recombinant genome having the genetic material of interest.
  • the recombinant genome in one instance includes genetic material encoding human parathyroid hormone (hPTH) .
  • hPTH human parathyroid hormone
  • it can also include a gene encoding a dominant selectable marker (e.g., the neo gene which encodes neomycin resistance in bacteria and G418 resistance in mammalian cells) .
  • the endothelial cells are transduced —that is, the genetic material of interest (in this case, DNA encoding hPTH and, optionally, the neo gene) is stably introduced into the endothelial cells.
  • the transduced endothelial cells express the encoded hPTH and, if the neo gene is present, also express it, resulting in cells having the selectable trait.
  • endothelial cells are transduced as a result of exposure to media containing infectious virus in which the recombinant genome includes genetic material of interest (e.g., any gene of interest or a portion thereof, RNA of interest) .
  • genetic material of interest e.g., any gene of interest or a portion thereof, RNA of interest
  • endothelial cells have been transduced with a gene which codes for a secreted 'product (human parathyroid hormone or hPTH) ; a gene which codes for a membrane receptor (low density lipoprotein receptor or LDLR) ; and a gene coding for an intracellular enzyme (beta-galactosidase) .
  • Endothelial cells expressing the incorporated genetic material are grown to confluence in tissue culture vessels; removed from the culture vessel in which they were grown; and introduced into or implanted into the body. They can be introduced into the recipient by one of several methods. For example, transduced endothelial cells can be seeded onto the lumen of a prosthetic vessel. The endothelial cells will then grow to confluence, covering the lumen of the vessel. They form the lining of what has been called a neointima (a well organized structure that resembles the inti a and media of a native vessel (i.e., a covering of endothelial cells with deeper layers of smooth muscle cells) .
  • a neointima a well organized structure that resembles the inti a and media of a native vessel (i.e., a covering of endothelial cells with deeper layers of smooth muscle cells
  • they can be grafted onto a blood vessel in vivo through the use of a catheter. It is also possible they can be introduced into body cavities which are lined by serosal membranes, such as the peritoneal cavity, the pleural space, and the pericardial space. In this case, the endothelial cells would seed the serosal lining and secrete the product into the cavity. The product would then be absorbed via the lymphatic system.
  • Another example which has been carried out is the implantation into dogs of vascular grafts that have been seeded with autologous retrovirus-transduced endothelial cells.
  • Replication-defective retroviruses with amphotrcpic host range have been used to stably introduce a reporter gene into the genome of the endothelial cells.
  • the lac Z gene has been used as the reporter gene because its product of expression, beta-galactosidase, can be detected in-situ through the use of enzyme histochemical assays that stain the cells cytoplasm blue.
  • External jugular veins harvested from adult mongrel dogs were used as a source of endothelial cells which were plated in vitro during a 10-14 day period by two serial passages.
  • Cells from each animal v/ere divided into two aliquots and were either infected with a replication defective retrovirus or mock infected.
  • Small diameter dacron grafts were seeded at subconfluent densities with endothelial cells by the autologous clot method and surgically implanted as carotid interposition grafts into the dog from which the cells were harvested; each dog received a graft seeded with the genetically modified cells and a contralateral graft seeded with MOCK infected cells. Five weeks after implantation, the grafts were harvested and analyzed.
  • Cells were enzymatically harvested from the luminal surface of a portion of the graft to permit a more detailed characterization.
  • Primary cultures of cells v/ere established and expanded in vitro for approximately 2-3 weeks prior to analysis.
  • Genetically modified endothelial cells were identified in this population by Southern analysis and by the cytochemical assay for viral-expressed beta-galactosidase.
  • the majority of cells harvested -from the graft and expanded in vitro retained differentiated endothelial function (less than 95%) .
  • the proportion of cells that expressed viral directed beta-galactosidase or contained proviral sequences was consistently decreased 2-10 fold when compared to the cultures that were analyzed at the time of seeding.
  • calf aorta is the source of endothelial cells, as was the case in the initial work describ'ed herein.
  • a typical protocol for isolation of endothelial cells from a large artery is described below. Sections of aortas freshly harvested are placed under sterile conditions,- in a solution containing collagenase (e.g., 0.5 mg/ml) for 15-20 minutes at 37°C.
  • tissue culture medium e.g., RPMI 1640
  • complete medium RPMI containing 15 mm Hepes, pH 7.4, penicillin/streptomycin and 20% fetal calf serum
  • the cells divide and eventually cover the plate; when the plate is confluent, the cells can be passaged using standard tissue culture techniques.
  • the purity of the cultures is assessed by uptake of fluorescent labeled acetylated LDL which is specifically taken up by endothelial cells. If the cultures are not pure (i.e., contaminated with cells other than endothelial cells, such as smooth muscle cells or fibroblasts) , endothelial cells are cloned by limiting dilution and expanded to yield pure cultures.
  • the life span of endothelial cells is limited in culture but varies markedly, depending on the anatomical source and donor animal. Bovine aortic endothelial cells have been maintained in culture for at least 15-20 passages.
  • Retroviruses are RNA viruses; that is, the viral genome is RN.A.
  • This genomic RNA is, however, reverse transcribed into a DNA intermediate v/hich is integrated very efficiently into the chromosomal DNA of infected cells.
  • This integrated DNA intermediate is referred to as a provirus.
  • the retroviral genome and the proviral DNA have three genes: the gag, the pol and the env, which are flanked by two long terminal repeat (LTR) sequences.
  • the gag gene encodes the internal structural (nucleocapsid) proteins; the pol gene encodes the RNA-directed DNA polymerase (reverse transcriptase) ; and the env gene encodes viral envelope glycoproteins.
  • the 5' and 3' LTRs serve to promote transcription and polyadenylation of virion RNAs.
  • Adjacent to the 5' LTR are sequences necessary for reverse transcription of the genome (the tRNA primer binding site) and for efficient encapsidation of viral RNA into particles (the Psi site).
  • Mulligan, R.C. In: Experimental Manipulation of Gene Expression, 11. Inouye (ed) , 155-173 (1983); Mann, R. , et al. , Cell, 33:153-159 (1983) ; Cone, R.D. and R.C. Mulligan, Proceedings of the National Academy of Sciences, U.S.A., 81:6349-6353 (1984) .
  • the Psi 2 cell line was constructed in the following manner: A mutant murine- leukemia virus (MuLV) genome in which the region or the genome implicated in the encapsidation of viral RNA into virions is deleted (the Psi sequence in Figure 1) was constructed. This genome was stably introduced in NIH3T3 cells by DNA cotransformation and stable transformants that produced all of the viral proteins used for encapsidation, yet budded noninfectious particles, were isolated.
  • MuLV murine- leukemia virus
  • the mutant of MuLV was constructed by deleting 351 nucleotides from an infectious proviral DNA clone between the putative env mRN4 5' splice site and the AUG that initiates the coding sequence for Pr 65 ⁇ ag .
  • the deletion was made from a Bal 1 site to a Pst 1 site and a Hind III site was generated at the point of deletion.
  • pMOV. (pMOVPsi " ) was constructed as follows: Three purified DNA fragments v/ere ligated together to construct pMOV Psi . The first was obtained by digesting pMOV Psi with Xho I to completion, followed by partial digestion with Eco RI. Chumakov, I. et al.
  • the fragment extending from the Xho I site at 2.0 U in MuLV, through the 3' LTR, 3' mouse flanking sequence, all of pBR322, and ending at the Eco RI site was purified from an agarose gel after electrophoretic separation. Vogelstein, B. and D. Gillespie, Proceedings of the National Academy of Sciences, USA, 761:615-619 (1979).
  • the second fragment was obtained by digestion of pMOV Psi with Bal I to completion followed by purification of the fragment extending from the Bal I site in pBR322 through 5' mouse flanking sequence and 5' LTR to the Bal I site located at 0.7 U of MuLV.
  • Hind III linkers (Collaborative Research) were then blunt-ligated to this fragment with T4 DNA ligase, and the fragment was digested with excess Hind III and Eco RI. The LTR-containing fragment was purified from an agarose gel after electrphoretic separation. The third fragment present in the final ligation reaction was obtained from pSV2-gag/pol v/here the gag/pol region of MuLV had been subcloned into pSV2. Mulligan, R.C. and P. Berg, Science, 209:1422-1427 (1980).
  • pSV2-gag/pol was digested to completion with Xho I and Hind III and the fragment extending from the Hind III site (changed from the Pst I site at 1.0 U of MuLV) to the Xho I site at 2.0 of MuLV was purified from an agarose gel following electrophoretic separation. These three DNA fragments were then mixed in equimolar amounts at a total DNA concentration of 50 ug/ml. in ligase buffer (50 mil Tris-HCl [pH 7.8], 10 mil MgC12, 20 mM dithiothreitol, 1.0 mil ATP, 50 ug/ml. bovine serum albumin) and incubated with T4 DNA ligase for 18 hr.
  • ligase buffer 50 mil Tris-HCl [pH 7.8], 10 mil MgC12, 20 mM dithiothreitol, 1.0 mil ATP, 50 ug/ml. bovine serum albumin
  • E. coli HB101 was transfected with the ligated DNA, and ampicillin resistant transfectant ⁇ were obtained.
  • the plasmid DNA obtained from a number of transformants was screened for the desired structure by digestion ! with appropriate restriction endonucleases and electrophoresis through agarose gels. Davis, R.W. et al. , Methods in Enzymology, 5:404-411 (1980).
  • pMOV-Psi The Psi 2 cell line described by M ⁇ illigan and co-workers was created by transfecting NIH 3T3 endothelial cells v/ith pMOV-Psi , which is an ecotropic Moloney murine leukemia virus (Mo-MuLV) clone.
  • Mo-MuLV Moloney murine leukemia virus
  • pMOV-Psi expresses all the viral gene products but lacks the Psi sequence, v/hich is necessary for encapsidation of the viral genome.
  • pMOV-Psi expresses an ecotropic viral envelope glycoprotein v/hich recognizes a receptor present only on mouse (and closely related rodent) cells.
  • Psi am line v/hich are Psi-2-like packaging cell lines.
  • These Psi-am cell lines contain a modified pMOV-Psi-genome, in which the eco ⁇ tropic envelope glycoprotein has been replaced with envelope sequences derived from the amphotropic virus 4070A. Hartley, J.W. and W.P. Rowe, Journal of Virology, l_9_:19-25 (1976). As a result, they are useful for production of recombinant virus with amphotropic host range.
  • the retrovirus used to make the Psi am cell line has a very broad mammalian host range (an amphotropic host range) and can be used to infect human cells.
  • the Psi-am cell line is capable of packaging recombina'nt retroviral genomes into infectious retroviral particles.
  • Cone, R. and Mulligan, R. Proceedings, of the National Academy of Sciences, USA, 8_1:6349-6353 (1984).
  • the retroviral genome has been modified by Cone and Mulligan for use as a vector capable of introducing new genes into cells. As shown in Figure 2, the gag, the pol and the env genes have all been removed and a DNA segment encoding the neo gene has been inserted in their place. The neo gene serves as a dominant selectable marker.
  • the retroviral sequence which remains part of the recombinant genome includes the LTRs, the tRNA binding site and the Psi packaging site. Cepko, C. et al. , Cell, 37:1053-1062 (1984) . Additional vector constructions which have been used in producing transduced endothelial cells of the present invention are represented in Figure 2 and are described in detail below. pLJ. The characteristics of this vector have been described in Korman, A.J. et al. , Proceedings of the National Academy of Sciences, USA 84:2150 (1987). This vector is capable of expressing two genes: the gene of interest and a dominant selectable marker, such as the Neo gene.
  • the gene of interest is cloned in direct orientation into a BamHI/Smal/Sall cloning site just distal to the 5' LTR, while, the Neo gene is placed distal to an internal promoter (from SV40) which is farther 3' than is the cloning site (is located 3' of the cloning site) .
  • Transcription from PLJ is initiated at two sites: 1) the 5' LTR, which is responsible for expression of the gene of interest and 2) the internal
  • SV40 promoter which is responsible for expression of the neo gene.
  • the structure of pLJ is represented in Figure 2a.
  • Vector pLJ is represented in Figure 2a.
  • the genetic material of interest is inserted just following the 5' LTR. Expression of this genetic material is transcribed from the LTR and expression of the neo gene is transcribed from an internal SV40 promoter.
  • pEm In this simple vector, the entire coding sequence for gag, pol and env of the wild type virus is replaced with the gene of interest, which is the only gene expressed. The components of the pEm vector are described below.
  • the 5' flanking sequence, 5 1 LTR and 400 bp of contiguous sequence (up to the BamHI site) is from pZIP.
  • the 3' flanking sequence and LTR are also from pZIP; however, the Clal site 150 bp upstream from the 3' LTR has been ligated with synthetic BamHI linkers and forms the other half of the BamHI cloning site present in the vector.
  • the Hindlll/EcoRl fragment of pBR322 forms the plasmid backbone.
  • This vector is derived from sequences cloned from a strain of Moloney Murine Leukemia virus.
  • An analogous vector has been constructed from sequences derived from the myeloproliferative sarcoma virus.
  • the structure of pEM is represented in Figure 2b. Vectors without a selectable marker can also be used to transduce endothelial cells with genetic material of interest.
  • Vectors are basically simplifications of the vectors previously described, in which there is such a marker.
  • Vector pEm is represented in Figure 2b; as represented, the main components of the vector are the 5' and 3' LTR, and the genetic material of interest, inserted between the two LTRs.
  • a cell line which produces recombinant amphotropic retrovirus having a recombinant genome is used to infect endothelial cells.
  • the recombinant genome can include a variety of components, but in general is comprised of two LTRs and, in place of the gag, the pol and the env sequences, a second promoter sequence. In some cases, it also includes a gene encoding a selectable marker (e.g. , neo) .
  • Viral stocks to be used in introducing genetic material of interest into endothelial cells, are harvested, as described above, supplemented with 8 micrograms per ml. (mcg/ml.) of Polybrene (Aldrich) and added to the culture of endothelial cells. If the titer of the virus is high (e.g., approximately 10 cfu per ml.) , then virtually all endothelial cells v/ill be infected and no selection (e.g., of endothelial cells into which the vector, including the recombinant genome, has been introduced) is required.
  • a retroviral vector that has a selectable marker, such as neo or his. If a selectable marker is used, after exposure to the virus, the cells are grown to confluence and split into selective media (e.g., media, containing G418 if the selectable marker is neo, media containing histidinol and no histidine if the selectable marker is his) .
  • the neo gene is a bacterial gene derived from the transposon Tn5, which encodes neomycin resistance in bacteria and resistance to the antibiotic G418 in mamma ⁇ lian cells.
  • This neo gene acts as a dominant'selectable marker; its presence in a mammalian cell converts the cell into one which will grow in the presence of G418. (If it is not present, the cell dies in the presence of G418.) As a result, the presence of this gene in a mammalian cell can be determined by culturing cells in media which contains G418.
  • the recombinant retrovirus having this recombinant genome is referred to as the neo virus.
  • the recombinant retroviral vectors having the neo gene also have a cloning site.
  • genetic material of interest can be introduced into the vector, incorporated into endothelial cells and expressed by endothelial cells transduced with the recombinant retrovirus (referred to as endothelial cells having incorporated genetic material) .
  • Retroviral vectors that express genes that encode three different classes of proteins have been constructed: a secreted hormone polypeptide (human PTH, or hPTH) , a membrane receptor (receptor for LDL, or LDLR) , and an intracellular enzyme (beta-galactosidase) . Efficient expression of the recombinant viruses when incorporated into endothelial cells has been demonstrated and is described in detail in the examples.
  • Genes encoding other proteins or polypeptides can also be introduced into endothelial cells by means of an appropriate retroviral vector.
  • a gene encoding human growth hormone (HGH) or a gene encoding insulin can be introduced into endothelial cells.
  • HGH human growth hormone
  • insulin can be introduced into endothelial cells, alone or in combination with a gene encoding a selectable marker, such as the neo gene.
  • HGH is a polypeptide of about 29,000 Daltons which is normally secreted only by the hypothalamus.
  • Insulin is a polypeptide which regulates glucose levels in the bloodstream.
  • genes, as well as others can be introduced into endothelial cells in the same manner as described above for the hPTH gene and the resulting transduced endothelial cells can be implanted into or applied to an appropriate site on the body.
  • the His D gene can be used for this purpose.
  • the His D gene is a bacterial gene from Salmonella and encodes histidinol dehydrogenase, a polypeptide which converts histidinol to histidine. Histidine is an essential amino acid; histi ⁇ dinol is an alcohol analogue of histidine and can be con ⁇ verted to histidine under the proper metabolic condi ⁇ tions. If cells are grown in media containing histidinol but lacking histidine, those cells having the His D gene can convert histidinol to histidine. Because histidine is essential to their function, those cells which have the His D gene (and thus can make histidine) will survive and those lacking the gene will not.
  • a retrovirus vector having the His D gene has been used to infect keratinocytes.
  • the keratinocytes contain ⁇ ing His D gene were selected by growing these cells in media lacking histidine but containing histidinol.
  • keratinocytes having the His D gene formed colonies and grew to confluence; those lacking the gene did not. In fact, such cells occurred at a much higher frequency than those in v/hich the neo gene was included.
  • SV40 SV40
  • herpes virus adenovirus
  • human papilloma virus can be used for this purpose.
  • vascular grafts or Implants Many important disease states involve stenosis or occlusion of arteries that supply vital organs.
  • the pathologic mechanism most commonly implicated in such disease states is atherosclerosis. Examples include angina pectoris and myocardial infarction due to coronary artery disease; transient ischemic attacks and strokes due to cerebral vascular disease; renal vascular hypertension, and ultimately, renal failure due to renal artery stenosis; and claudication of the lower extremities, which is caused by vascular disease of peripheral arteries and, in its most severe form, can result in amputation.
  • the agents that predispose an individual to atherosclerotic lesions are eliminated (e.g., hypertension, cigarette smoking, and hypertension)
  • the natural history of these disease states is usually progression of the atherosclerotic lesions, resulting in permanent damage or death.
  • An accepted and widely used therapeutic approach to advanced atherosclerotic disease is to bypass the site of major stenosis or occlusion with a prosthetic vessel made of synthetic material, such as Dacron. More than 350,000 vascular grafts are implanted each year.
  • One major problem with this approach is that the prosthetic vessel is extremely thrombogenic (i.e., it has the propensity to develop clots) , which leads to a very high rate of re-stenosis. It has been possible to reduce this problem by seeding the lumen of the prosthetic vessels with autologous endothelial cells; grafts lined with endothelial cells are presumably less thrombogenic. It is in this setting that modified endothelial cells would be particularly useful.
  • Endothelial cells can be genetically modified according to the method of the present invention to improve their performance in the context of an endothelial cell-lined prosthetic implant.
  • Existing protocols for using endothelial cell-lined prosthetic implants are complicated by several significant technological problems, most of which can be overcome through the use of genetically engineered endothelial cells.
  • endothelial cells can be genetically modified to enhance their proliferative capacity by infecting them with a retrovirus which expresses an endothelial cell mitogen, such as endothelial cell growth factor. It may be possible to initially seed an implant with transduced endothelial cells at subconfluent densities and subsequently achieve a confluent lining in vitro or in vivo. Many applications of endothelial cell-lined prosthetic implantation which have been impossible due to limited quantities of autologous cells may now be feasible.
  • the modified endothelial cells may exhibit a selective advantage over competing cells (such as smooth muscle cells or endogenous endothelial cells) , thereby preventing overgrowth of the transduced endothelial cells by the competing cells.
  • An additional problem with endothelialized implants is that the lumen of the prosthetic vessel undergoes progressive narrowing due to the proliferation of smooth muscle cells between the wall of the prosthesis and the luminal surface.
  • One way in which this can be prevented is to introduce into the endothelial cells a gene that secretes a product which inhibits the growth of smooth muscle cells.
  • Many types of autocrine:paracrine growth factors that control proliferation of mesenchymal and/or epithelial cells have recently been identified and their genes cloned. Such genes can be introduced into endothelial cells using the method of the present invention. The resulting transduced endothelial cells produce the cell growth inhibitor.
  • a further technical problem of endothelialized implant protocols is that the binding (plating) efficiency of the endothelial cells to the prosthetic graft is relatively low. Previous attempts to improve this have been directed at modifying the composition of the graft surface and have been of limited success.
  • Using the method of the present invention it is possible to introduce into endothelial cells a gene which codes for a membrane receptor.
  • the lumen of the prosthetic vessel can then be coated with the ligand for the receptor, thereby facilitating binding of endothelial cells to the luminal surface through the membrane receptor/ligand interaction.
  • Genetically engineered endothelial cells of the present invention can be used to decrease the thromJoogenicity of endothelial cell-lined prosthetic grafts.
  • the mechanism of clot formation is believed to be platelet adhesion follov/ed by deposition of fibrin and propagation of clot. This could be minimized or possibly eliminated by seeding the grafts with genetically engineered endothelial cells that secrete a thrombolytic agent (e.g., an agent which dissolves clots, such as tissue plasminogen activator (TPA) or streptokinase) .
  • TPA tissue plasminogen activator
  • streptokinase streptokinase
  • Modified Endothelial Cells to Deliver a Product to a Limb or Organ
  • This invention can also be used to introduce into endothelial cells genes that secrete factors which would be beneficial to the limb or organ perfused by the artery containing the prosthesis.
  • a common clinical problem is the presence of extensive narrowing of small vessels distal to the site of the prosthetic vessel. This is characteristic of the vascular disease associated with diabetes mellitus. Revascularization of the larger vessels with implants leads to incomplete reconstitution of perfusion , to the affected limb or- organ.
  • endothelial cells in the affected area secrete a factor or factors that promote angiogenesis.
  • the genes for many of these angiogenic factors have been cloned and can be introduced into endothelial cells (e.g. , using a retrovirus in which the genome includes the gene of interest).
  • the delivery of the angiogenic factor(s) would be targeted to the organ or limb supplied by the artery containing the graft.
  • the effective concentration of the factor would be relatively high in the affected tissue, but low in general circulation because it would be diluted in the venous circulation upon its return to the heart.
  • vasodilator Another way of further promoting vascular flow to a compromised organ or limb is to maximally dilate all afferent vessels. Attempts to do this with oral or parenteral medicines have resulted in little therapeutic benefit, accompanied by many systemic side effects. This is caused, in part, by the fact that the vasodilator is not targeted to the appropriate tissue. Endothelial cells engineered to secrete a potent vasodilator such as atrial naturetic factor is an alternative approach. In this application, the affected organ or limb can be perfused with arterial blood which contains a very high concentration of a vasodilator. This results in an increase in the overall vascular perfusion. However, the vasodilator is diluted to nonpharmacologic levels upon return to the heart, thereby obviating the many systemic side effects of vasodilators that occur when administered in a nonselective manner.
  • the present invention makes it possible to geneti ⁇ cally engineer endothelial cells in such a manner that they produce a selected protein or polypeptide, such as selected polypeptides and proteins not normally produced in endothelial cells in biologically significant amounts, and secrete them into the bloodstream or other area of the body (e.g., the central nervous system).
  • the endothelial cells formed in this way can serve as a continuous drug delivery system to replace present regimens, which require periodic administration (by ingestion, injection, etc.) of the needed substance.
  • insulin can be used to provide continuous delivery of insulin, which, at the present time, must be isolated from the pancreas, extensively purified and then injected into the body by those whose insulin production or utilization is impaired. In this way, insulin can be introduced into the body via a continuous drug delivery system and, as a result, there would be no need for daily injections of insulin.
  • Endothelial cells can also be used for the production of clotting factors. Hemophiliacs lack a protein called Factor VIII, which is involved in clotting. Factor VIII is now administered by injection. Endothelial cells having genes encoding Factor VIII can be. used to make an implant in which they produce Factor VIII; as an implant, the tissue would secrete the factor into the bloodstream. Incorporation of genetic material of -interest into endothelial cells can be particularly valuable in the treatment of inherited disease and the treatment of acquired disease. In the case of inherited diseases, this approach is used to provide genetically modified endothelial cells and other cells which can be used as a metabolic sink.
  • endothelial cells would serve to degrade a potentially toxic substance.
  • transduced endothelial cells used in treating urea cycle disorders can also be used in the treatment of genetic diseases in which a product (e.g. , an enzyme or hormone) normally produced by the body is not produced or is made in insufficient quantities.
  • a product e.g. , an enzyme or hormone
  • endothelial cells transduced with a gene encoding the missing or inadequately produced substance can be used to produce it in sufficient quantities. For example, this can be used in producing alpha-1 anitrypsin. It can also be used in the production of Factor VIII and Factor IX, and thus would be useful in treating hemophilia.
  • endothelial cells i.e., endothelial cells transduced with genetic material of interest
  • endothelial cells i.e., endothelial cells transduced with genetic material of interest
  • endothelial cells can be used in treating anemia, which is commonly present in chronic disease and often associated with chronic renal failure (e.g., in hemodialysis patients).
  • endothelial cells having incorporated in them a gene encoding erythropoietin would correct the anemia by stimulating the bone marrow to increase erythropoiesis (i.e. production of red blood cells).
  • Endothelial cells of the present invention can also be used to administer a low dose of tissue plasminogen activator as an activator to prevent the formation of thrombi.
  • endothelial cells having incorporated genetic material which encodes TPA would be transplanted into an individual in whom thrombus prevention is desired. This would be useful, for example, as a prophylactic against common disorders such as coronary artery disease, cerebrovascular disease, peripheral vascular occlusive disease, vein (e.g., superficial) thrombosis, such as seen in pulmonary emboli, or deep vein thrombosis.
  • Endothelial cells which contain DNA encoding calcitonin can be used in the treatment of Paget's Disease, a progressive, chronic disorder of bone metabolism.
  • Endothelial cells engineered to produce and secrete interleukins can be used in several contexts.
  • the result of some of the therapies now used e.g., chemotherapy
  • neutropenia the presence of abnormally low numbers of neutrophils in the blood
  • use of virtually all the chemotherapeutic agents, as well as AZT, used in the treatment of (AIDS) Acquired Immune Deficiency Syndrome results in neutropenia. This condition results in numerous life-threatening infections.
  • administration of, for example, IL-3 through implantation of endothelial cells v/hich contain genetic material encoding IL-3 can be used to increase the neutrophil count.
  • administration of thrombopoietin, which stimulates the production of platelets can be used in the treatment of numerous conditions in which platelet count is low.
  • endothelial cells transduced with the gene for thrombopoietin can be applied to/implanted in an individual; production and secretion of the encoded product will result in stimulation of platelet production.
  • endothelial cells having incorporated genetic material are potentially valuable in the treatment of AIDS.
  • Interleukin 2 and Interleukin 3 which stimulate the immune system, are potentially valuable in the treatment of AIDS. They could be delivered by a vessel implant having endothelial cells v/hich have been genetically engineered to produce these two polypeptides (v/hich are now administered by periodic injection) .
  • Another use of the present invention is in the treatment of enzyme defect diseases. In this case the product encoded by the gene introduced into endothelial cells is not secreted (as are hormones) ; rather, it is an enzyme which remains inside the cell.
  • the present invention also has veterinary applica ⁇ tions. It can be used, for example, in delivering substances such as drugs (e.g., antibiotics) and hormones to animals, which would otherwise be provided by being incorporated into their feed, added to their water or injected periodically (e.g., daily or less frequently).
  • drugs e.g., antibiotics
  • hormones e.g., hormones
  • Use of the modified endothelial cells of the present invention has the advantage that the tissue formed of the modified cells can be applied to the animal and will provide quantities of the encoded protein on an ongoing basis, thus eliminating the need for daily/periodic administration of the substance.
  • genetic material of interest can be inserted at the BamHI cloning site of pLJ.
  • the genetic material of interest can be DNA as described above.
  • hPTH human parathyroid hormone
  • Equal quantities of the pLJ linear backbone and the BamHI PTH fragment were added together, in the presence of T4 DNA ligase.
  • the resulting mixture was maintained under conditions appropriate for ligation of the two fragments.
  • the ligation mixture was used to transform bacteria HB101 , v/hich were then plated onto agar containing kanamycin.
  • Maniatis, T. et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, p.p. 250-251, 504; Bolivar, F. and K.
  • Parathyroid hormone is a polypeptide which has a role in the regulation of calcium in the body.- Although the hPTH gene is present in human endothelial cells, it is not expressed in those cells at biologically significant levels. Endothelial cells capable of making a polypeptide hormone such as hPTH, or another substance not normally made by such cells at biologically significant levels, can be engrafted onto or implanted into an individual and serve as a continuous supply system for the hormone, or other substance.
  • the Psi am cells producing the recombinant virus construct which contained the hPTH-encoding DNA and DNA encoding a selectable marker (such as the neo gene) were used to produce a viral stock, as described above.
  • the viral stock was harvested; endothelial cells to be transduced with the virus containing the hPTH gene were incubated with the stock.
  • a selectable marker is used to identify and select for transduced endothelial cells by culturing on media containing G418. If the viral titer is sufficiently high, essentially all endothelial cells are infected and selection, using a selectable marker and appropriate media, is not needed.
  • endothelial cells transduced with the recombinant retrovirus having the hPTH gene to express the hPTH gene has been assessed _in vitro as follows: The in vitro production of hPTH by endothelial cells established from a bovine aorta and infected with a virus containing the hPTH gene was assessed.
  • the bovine aortic endothelial cells were derived from an explant from the aorta of a calf. The cells have a limited life span and are referred to as secondary cultures.
  • Bovine aortic endothelial cells were infected with virus and not selected in neomycin, as described above. Transduced bovine aortic endothelial cells were seeded onto 10 cm tissue culture dishes and grown to confluence. -Fresh culture media (DME with 10% CS and penicillin and streptomycin) was then added; this point is referred to subsequently as time zero. At the end of 24 hours, the media was removed and the cells were assayed for the production of human PTH.
  • DME DME with 10% CS and penicillin and streptomycin
  • the assay has a sensitivity of approximately one nanogram/milliliter serum (ng/ml) and was shown to be specific for human PTH in that it does not cross react with calf PTH.
  • the results of the experiments are reported as the production of hPTH, as measured by RIA, over time. Results are shown in Table I.
  • BAE bovine aortic endothelial
  • Endothelial cells from a bovine aorta transduced with DNA encoding hPTH according to the method of the present invention have been deposited at the American Type Culture Collection (Rockville, MD) under deposit number CRL9601, under conditions appropriate to meet the requirements of the Budapest Treaty.
  • the cDNA for human LDL receptor (LDLR) was inserted into the pEM vector.
  • the cDNA for LDLR was prepared for insertion into the pEM as follows.
  • LDLR cDNA was excised from the vector pTZl (obtained from Dr. Goldstein, University of Texas Health Science Center) by digestion with Hindlll.
  • the 2.6 kb Hindlll fragment containing all of the LDLR coding sequence v/as blunt ended with lenow fragment and Bell oligonucleotide linkers (from NEB) were ligated to the cDNA with T4 DNA ligase.
  • this ligation mixture was digested with an excess of the enzyme Bell and the fragment was fractionated on an agarose gel and purified.
  • pEM was digested with BamHI and the linearized plasmid was digested with calf intestinal phosphatase. Equal quantities of the linkered LDLR insert and pEM backbone were mixed and ligated with T4 DNA ligase. The ligation mixture was used to -transform HBlOl and ampicillin resistant colonies were analyzed for the appropriate retroviral vector.
  • the pEM-LDLR vector was transfected into the Psi-am cell line and clones that produced high quantity of recombinant virus were identified. Viral stocks were used to infect cultures of bovine aortic endothelial cells as described for PTH.
  • LDLR expression was carried out as follows: confluent plates of control or transduced bovine aortic endothelial cells were incubated with LDL that had been conjugated with a fluorescent label (obtained from Biomedical Tech Inc., Stoughton, MA) at a concentration of 10 ug/ml for approximately 8 hours. Following this, the cells were washed with PBS and fixed in 0.5% gluteraldehyde in PBS. They were then visualized under a fluorescent microscope for the uptake of fluorescent labeled LDL (*LDL) . The results of this experiment are presented in Figure 4.
  • a fluorescent label obtained from Biomedical Tech Inc., Stoughton, MA
  • the level of endogenous LDLR is low, as evidenced by the relative lack of *LDL uptake in uninfected cultures (See 4B) .
  • the level of endogenous LDLR is low, as evidenced by the relative lack of *LDL uptake in uninfected cultures (See 4B) .
  • approximately 30% of all cells take up detectable quantities of *LDL, thereby documenting efficient expression of the exogenous LDLR (See 4D) .
  • the gene endocing beta-galactosidase from E. coli was inserted into pLJ and this vector was transfected into the Psi-am cell line, resulting in production of high titer retroviral stocks.
  • the construction of this vector and isolation of the producer cell line has been described by Price and co-workers. Price, J. et al. , Proceedings of the National Academy of Sciences, USA, 8_4:156-160 (1987). Stocks of virus encoding the beta-galatosidase gene were used to infect bovine aortic endothelial cells, as described earlier. The infected cultures were analyzed for beta-galatosidase expression using an in situ histochemical stain. See Price et al. above.
  • Endothelial cells were enzymatically harvested from external jugular veins of adult mongrel dogs which weighed 20-25 kg. Cells from each animal were divided into 2 aliquots; one to be infected with a replication defective retrovirus (see below) and the other to be mock infected. The ' enzymatically harvested cells were used to establish primary cultures. Endothelial cells were plated on to fibronectin coated flasks and maintained in 11199 Medium supplemented with 5% plasma derived equine serum, penicillin, streptomycin, heparin and ECGF during two serial passages over a 10-14 day period.
  • Replication-defective retroviruses with amphotropic host range were used to stably introduce a reporter gene into the genomic DNA of the endothelial cells.
  • the lac Z gene was used as the reporter gene because its product of expression, beta-galactosidase, can be detected in situ through the use of enzyme histochemical assays that stain the cell's cytoplasm blue. (Example 3).
  • Efficiency of retroviral infection was estimated by Southern analysis which detects proviral sequences and by the cytochemical stain for viral-expressed beta-galactosidase which detects infected cells in situ. There were two recombinant retroviruses used in these studies.
  • the BAG vector has been described previously by Price et al.
  • the EA-G virus containing the Lac Z-gene, expresses Beta-galactosidase from the 5' LTR (Long Terminal Repeat) and a selectable marker (Neo) from an SV40 derived pro ⁇ moter which confers resistance to kanamycin in prokaryotes and G418 in eukaryotes.
  • a selectable marker from an SV40 derived pro ⁇ moter which confers resistance to kanamycin in prokaryotes and G418 in eukaryotes.
  • Approximately 5-15% of the endothelial cells exposed to the BAG virus were infected (summarized in Table II) ; cultivation of these cultures in media supplemented with the aminoglycoside G418 effectively selected for transduced cells.
  • the BAL vector is derived from the previously described BA-LDLR vector except LDLR cDNA sequences were replaced with the coding sequences for the E. coli beta-galactosidase gene.
  • the higher titer BAL virus, containing the Lac Z-gene, expresses beta-galactosidase from a promoter derived from the chicken beta-actin. Approximately 50% of the cells exposed to the BAL virus were transduced as measured by Southern analysis and by the in situ cytochemical stain for beta-galactosidase.
  • Retrovirus-infected endothelial cells were analyzed at the time of seeding and after removal from the implanted graft for uptake of DIL-AC-LDL and for expression of viral-directed beta-galactosidase.
  • Pre-seeded and post-implantation cells that had been inoculated with DIL-AC-LDL were assessed using phase contrast and fluorescent micrographs.
  • Pre-seeded and post-implanted cells were also analyzed for the expression of viral directed beta-galactosidase.
  • the cultures were analyzed for endothelial cell specific function (i.e., uptake of acetylated LDL, and the presence of Von Willebrands factor) and for the presence of antigens specific for smooth muscle cells. These analyses indicated that more than 98% of the cells from each isolate were functioning endothelial cells.
  • the experimental system was a modification of a previously described dog model that has been used to study graft repopulation. Dogs 1-3 received implants that had been seeded with BAG-infected, unselected endothelial cells while dogs 4-7 initially received implants seeded with the BAL-infected, unselected endothelial -cells.
  • endothelial cells from dogs 4-7 were also infected with the BAG virus and expanded in culture in the presence or absence of G418, an aminoglycoside that selects for BAG-transduced cells.
  • the grafts were explanted for analysis and new grafts were implanted in dogs 4-7 (referred to as dogs 4'-7').
  • dogs 4'-7' Each of these dogs received a graft seeded with endothelial cells that were infected with BAG virus and selected in G418 and a contralateral graft seeded with BAG infected unselected endothelial cells.
  • the second set of grafts were again explanted after 5 weeks.
  • Animals 4" through 7 1 represent the second experiment performed on animals 4 through 7 (see text) .
  • BAG-U represents BAG infected endothelial cells unselected with G418 for transduced cells.
  • BAG-S represents BAG infected endothelial cells selected with G418 for transduced cells.
  • BAL represents BAL infected endothelial cells.
  • MOCK represents MOCK infected cells.
  • E is an indication of the efficiency of infection as measured by the beta-galactosidase stain. Patency of the graft after 5 weeks is indicated with a (Y) yes or (N) no. Coverage of the graft is an indication of the per cent of the surface repopulated as measured by scanning electron microscopy.
  • Grafts were cut longitudinally and a portion fixed in 0.5% gluteraldehyde for 5 minutes, washed in PBS three times, and incubated in x-gal solution for 2 hours and the luminal surface photographed with a Leitz dissecting microscope. The grafts v/ere visualized enface and several interesting aspects of the seeding pattern were observed. The density of transduced cells was greatest . in the deeper surfaces of the crimped graft. This was visualized under low power as rings of blue staining cells that line the crevices of the serrated surface and correlates with the variation in surface endothelial cell repopulation visualized by scanning electron microscopy.
  • Cells were enzymatically harvested from the luminal surface of portions of the grafts to permit a more detailed characterization.
  • Primary cultures of cells were established and expanded in vitro for approximately 2-3 weeks prior to analysis.
  • Genetically modified endothelial cells were identified in this population of cells by Southern analysis and by the cytochemical assay for viral-expressed beta-galactosidase.
  • the majority of cells harvested from the graft and expanded in vitro retained differentiated endothelial function (less than 95%) .
  • the proportion of cells that expressed viral directed beta-galactosidase or contained proviral sequences was consistently diminished when compared to the cultures analyzed at the time of seeding. This disparity is due in part to the partial repopulation of grafts with endogenous cells by growth through interstices or from the anastomoses.
  • This invention has indistrial applicability in improving performance (e.g., duration, patency, thrombogenicity) of vascular grafts or implants, made of synthetic materials, which are often used in treating advanced atherosclerotic disease. It is also useful in providing hormones, enzymes and drugs to mammals, includ ⁇ ing humans, in need of such substances. For example, it can be used to provide a continuous supply of a hormone which otherwise would be administered on a intravenously, intramuscularly or subcutaneously. It is particularly valuable in providing such substances which are needed for extended periods of time.

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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Cellules endothéliales dans lesquelles a été incorporée une matière génétique codant pour un polypeptide ou une protéine d'intérêt et facultativement, un marqueur sélectionnable, et procédés permettant de fabriquer et d'utiliser les cellules endothéliales ainsi traitées. De telles cellules endothéliales sont utiles pour améliorer l'efficacité de greffes vasculaires ainsi que pour administrer le polypeptide ou la protéine codé tel qu'une enzyme, une hormone ou un médicament, à un individu ayant reçu une greffe ou un implant desdites cellules.
PCT/US1988/004383 1987-12-11 1988-12-08 Modification genetique de cellules endotheliales Ceased WO1989005345A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP89900771A EP0391960B1 (fr) 1987-12-11 1988-12-08 Modification genetique de cellules endotheliales
DE3851153T DE3851153T2 (de) 1987-12-11 1988-12-08 Genetische modifizierung von endothelialen zellen.
HK98107130A HK1008400A1 (en) 1987-12-11 1988-12-08 Genetic modification of endothelial cells

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US13192687A 1987-12-11 1987-12-11
US131,926 1987-12-11

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WO1989005345A1 true WO1989005345A1 (fr) 1989-06-15

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PCT/US1988/004383 Ceased WO1989005345A1 (fr) 1987-12-11 1988-12-08 Modification genetique de cellules endotheliales

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EP (1) EP0391960B1 (fr)
JP (1) JP2914692B2 (fr)
AT (1) ATE110108T1 (fr)
CA (1) CA1340427C (fr)
DE (1) DE3851153T2 (fr)
HK (1) HK1008400A1 (fr)
WO (1) WO1989005345A1 (fr)

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DE3851153D1 (de) 1994-09-22
JPH03505036A (ja) 1991-11-07
CA1340427C (fr) 1999-03-09
EP0391960A1 (fr) 1990-10-17
EP0391960B1 (fr) 1994-08-17
JP2914692B2 (ja) 1999-07-05
HK1008400A1 (en) 1999-05-07
DE3851153T2 (de) 1995-01-05
ATE110108T1 (de) 1994-09-15

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